Although protein medicines have become more common with the advent of recombinant DNA technology, the pharmaceutical industry still prefers the more traditional small molecule drugs because of the poor pharmacokinetic and other properties (such as absorption, distribution, metabolism and excretion) of proteins as exogenously-administered therapeutic agents. Using proteins as therapeutic drugs, rather than small molecule mimics of these proteins, is certainly more natural and might be preferred if the aforementioned problems can be overcome.
The main problem is that proteins cannot be given orally since they are digested in the gastrointestinal tract. Even if the digestive process is suppressed, proteins alone still cannot transit from the lumen across the epithelial cell barrier into the bloodstream. Intramuscular or subcutaneous injection is most commonly used. Even so, most proteins have a very short half-life (measured in minutes), so the injected protein is present in the patient for only brief periods of time. A solution to this problem would be the development of orally-bioavailable protein drugs. The natural slowness of the digestive process would spread the absorption of the protein drug into the bloodstream over one or more hours, and there would be no significant obstacle to taking a pill several times each day.
The therapeutic efficacy of an orally-administered drug is dictated not only by its pharmacological properties such as potency and selectivity, but also by its biopharmaceutical characteristics such as membrane permeability and metabolic stability. In the past decade, several in-vitro and in-vivo screening techniques have been developed to assess intestinal membrane permeability of therapeutic agents as an indicator of oral absorption (e.g., B. H. Stewart, O. H. Chan, N. Jezyk, and D. Fleisher, 1997, Discrimination between drug candidates using models for evaluation of intestinal absorption, Adv. Drug Del. Res. 23:27-45). The rate of intestinal absorption of a compound is critically influenced by its physicochemical properties, which in turn is dependent on its structural features. Thus, in order to gain insight into the processes involved in the intestinal transport of compounds, elucidation of solute structure/permeability relationships is essential.
The successful oral delivery of peptides and peptidomimetics poses numerous challenges. Low permeability, lack of proteolytic stability, and binding to intestinal components are some of the main factors leading to their low oral bioavailability. The proton linked intestinal oligopeptide transporter (PepT1) facilitates the apical transport of smaller peptides (i.e., typically less than 4 amino acid residues) and some peptide-like drugs (P. V. Balimane, I. Tamai, A. Guo, T. Nakanishi, H. Kitada, F. H. Leibach, A. Tsuji, and P. J. Sinko. Direct evidence for a peptide transporter (PepT1)-mediated uptake of a nonpeptide prodrug valacyclovir, Biochem. Biophys. Res. Commun. 250:246-251 (1998); A. Tsuji and I. Tamai. Carrier-mediated intestinal transport of drugs, Pharm. Res. 13:963-977 (1996); A. Tsuji, I. Tamai, H. Hirooka and T. Terasaki. Beta-lactam antibiotics and transport via the dipeptide carrier system across the intestinal brush-border membrane, Biochem. Pharmacol. 36:565-567 (1987); P. J. Sinko and G. L. Amidon. Characterization of the oral absorption of beta-lactam antibiotics. II. Competitive absorption and peptide carrier specificity, J. Pharm. Sci. 78:723-727 (1989)). PepT1 is a low affinity, high capacity transporter that is involved with the absorption of relatively large doses (i.e., milligram quantities) of drugs such as the cephalosporins and penicillin antibiotics (P. J. Sinko and G. L. Amidon. Characterization of the oral absorption of beta-lactam antibiotics. II. Competitive absorption and peptide carrier specificity, J. Pharm. Sci. 78:723-727 (1989)). Larger peptides such as Leu-enkephalin, a pentapeptide, are not substrates for PepT1 and, therefore, are relatively poorly absorbed (R. T. Borchardt. Optimizing oral absorption of peptides using prodrug strategies. J. Controlled Rel. 62:231-8 (1999)). It is possible to enhance the oral absorption of low permeability, larger peptides by enhancing their stability to proteolytic degradation in the gastrointestinal (GI) tract (D. I. Friedman and G. L. Amidon. Oral absorption of peptides: Influence of pH and inhibitors on the intestinal hydrolysis of leu-enkephalin and analogues, Pharm. Res. 8:93-96 (1991); J. P. Bai, L. L. Chang, and J. H. Guo. Effects of polyacrylic polymers on the luminal proteolysis of peptide drugs in the colon, J. Pharm. Sci. 84:1291-1294 (1995); J. P. Bai, L. L. Chang, and J. H. Guo. Effects of polyacrylic polymers on the degradation of insulin and peptide drugs by chymotrypsin and trypsin, J. Pharm. Pharmacol. 48:17-21 (1996)). However, net peptide absorption remains relatively low if the effective permeability across the intestinal mucosa is also not enhanced. Using citric acid to reduce intestinal pH and minimize trypsin activity and lauroyl carnitine to enhance permeability, a significant enhancement in the oral bioavailability of a large peptide, salmon calcitonin, was achieved (Y-H. Lee, B. A. Perry, S. Labruno, H. S. Lee, W. Stern, L. M. Falzone, and P. J. Sinko. Impact of regional intestinal pH modulation on absorption of peptide drugs: Oral absorption studies of salmon calcitonin in beagle dogs, Pharm. Res. 16(8):1233-1239 (1999); P. J. Sinko, Y-H. Lee, V. Makhey, G. D. Leesman, J. P. Sutyak, H. Yu, B. Perry, C. L. Smith, P. Hu, E. J. Wagner, L. M. Falzone, L. T. McWhorter, J. P. Gilligan, and W. Stern. Biopharmaceutical approaches for developing and assessing oral peptide delivery strategies and systems: In Vitro permeability and In Vivo oral absorption of salmon calcitonin (sCT), Pharm. Res. 16(4):527-533 (1999); P. J. Sinko, C. L. Smith, L. T. McWhorter, W. Stern, E. Wagner, and J. P. Gilligan. Utility of pharmacodynamic measures for assessing the oral bioavailability of peptides. 1. Administration of recombinant salmon calcitonin in rats, J. Pharm. Sci. 84(11): 1374-1378 (1995)).
Another common strategy for improving the intestinal permeability of poorly absorbed compounds is the use of permeation enhancers that transiently modify the barrier properties of biological membranes. Despite initial enthusiasm, the invasive nature of this approach and its associated side-effects have severely hampered the use of absorption enhancers as a viable strategy for improving intestinal permeability (Hochman, J.; Artursson, P. Mechanisms of absorption enhancement and tight junction regulation. J. Controlled Release 1994, 29, 253-267. Citi, S.; Protein kinase inhibitors prevent junction dissociation induced by low extracellular calcium in MDCK epithelial cells. J. Cell Biol. 1992, 117(1), 169-178). Newer agents such as zonulin (Fasano, A.; Novel approaches for oral delivery of macromolecules. J. Pharm. Sci. 1998, 87(11), 1351-1356; Fasano, A. Modulation of intestinal permeability: An innovative method of oral drug delivery for the treatment of inherited and acquired human diseases. Mol Gen. Metabolism 1998, 64, 12-18), that act by receptor-mediated, regio-specific and reversible mechanisms displaying considerably lower cytotoxicity and systemic side-effects, now offer a promising tool in permeability enhancement. However, further studies are still necessary to fully establish their therapeutic utility.
An alternative, non-invasive approach to facilitate intestinal drug absorption is to target specific absorptive transporter systems by chemical modification of drugs to prodrugs and analogues. For instance, it has previously been demonstrated that unlike acyclovir (an anti-herpetic nucleoside), its L-valyl ester prodrug, valacyclovir, is a substrate of the intestinal proton-linked oligopeptide transporter, PepT1 (Guo, A.; Hu, P.; Balimane, P. V.; Leibach, F. H.; Sinko, P. J. Interactions of a nonpeptidic drug, valacyclovir, with the human intestinal peptide transporter (hPepT1) expressed in a mammalian cell line. J. Pharmacol. Exp. Ther. 1999, 289, 448-454; Balimane, P. V.; Tamai, I.; Guo, A.; Nakanishi T.; Kitada, H.; Leibach, F. H.; Tsuji, A.; Sinko, P. J. Direct evidence for peptide transporter (PepT1)-mediated uptake of a nonpeptide prodrug, valacyclovir. Biochem. Biophys. Res. Commun. 1998, 250, 246-251). Due to the low affinity, high capacity nature of PepT1, the interaction between valacyclovir and PepT1 results in a three to four-fold increase in the bioavailability of acyclovir. Despite accepting a wide range of endogenous and exogenous substrates with peptide-like structures, PepT1, facilitates the apical transport of only di- and tri-peptides, which makes it an unsuitable target for transporting larger peptides (>5 amino acid residues) across the intestine (Amidon, G. L.; Lee, H. J. Absorption of peptide and peptidomimetic drugs. Annu. Rev. Pharmacol. Toxicol. 1994, 34, 321-341., Ganaphthy, V.; Leibach, F. H.; Expression and regulation of the taurine transporter in cultured cell lines of human origin. Adv. Exp. Med. Biol. 1994, 359, 51-57.). However, like most currently used strategies for enhancing peptide absorption, it is nonspecific or the mechanisms of action are unknown making it difficult to precisely control the resulting in vivo effect.
The foregoing comments have their counterparts in transport across the blood-brain (and other related) barriers in which endothelial cell tight junctions gate the transport from the lumen of the capillary into the tissue or organ. Various obstacles to the transport of compounds are known and impact the availability of central nervous system active agents to those with the ability to translocate across the capillary endothelium or disrupt the intercellular connections.
The advent of combinatorial chemistry has facilitated potential correlations between intestinal absorption of congeneric series of compounds and iteratively designed newer compounds and their physicochemical properties. Several groups have tried to correlate the Caco-2 cell monolayer permeability of candidate compounds with their structural attributes derived using computational techniques. Parameters such as hydrogen bonding potential, solute lipophilicity, size, charge, and conformation have been shown to be important descriptors of intestinal transport (see, for example, K. Palm, K. Luthman, A-L. Ungell, G. Strandlund, and P. Artursson, Correlation of drug absorption with molecular surface properties, J. Pharm. Sci. 85 (1996) 32-39; C. A. Lipinski, F. Lombardo, B. W. Dominy, and P. J. Feeney, Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings, Adv. Drug Del. Res. 23 (1997) 3-25; O. S. Gudmundsson, S. D. S Jois, D. G. Vander Velde, T. J. Siahaan, B. Wang, R. T. Borchardt, The effect of conformation on the membrane permeation of coumarinic acid- and phenylpropionic acid-based cyclic prodrugs of opioid peptides, J. Peptide Res. 53 (1999) 383-392; K. Palm, K. Luthman, A-L. Ungell, G. Strandlund, F. Beigi, P. Lundahl, and P. Artursson, Evaluation of dynamic polar molecular surface area as predictor of drug absorption: Comparison with other computational and experimental predictors, J. Med. Chem. 41 (1998) 5382-5392; J. T. Goodwin, B. Mao, T. J. Vidmar, R. A. Conradi, and P. J. Burton, Strategies toward predicting peptide cellular permeability from computed molecular descriptors, J. Peptide Res. 53 (1999) 355-369; and E. G. Chikhale, K-Y. Ng, P. S. Burton, and R. T. Borchardt, Hydrogen bonding potential as a determinant of the in vitro and in situ blood-brain barrier permeability of peptides, Pharm. Res. 11 (1994) 412-419.). Conventional structure-transport analyses have only explored paracellular and passive transcellular routes of diffusion. Very little progress has been made in our understanding on the role of structural descriptors in transporter-mediated absorption processes, primarily due to the non-availability of 3-dimensional structure of membrane transporters.
With regard to the blood-brain barrier (BBB), in vitro models of BBB, the physiological interface regulating drug entry to the central nervous system (CNS), can mimic its in vivo barrier properties. These models offer an alternative to in vivo or in situ animal studies for the evaluation drug transport across the cell monolayers.
In co-pending application Ser. No. 09/044,411, now U.S. Pat. Nos. 6,258,774 and 60/267,396, disulfide-linked conjugates of polymers and therapeutic agents were prepared and shown to enhance intracellular delivery, particularly when a cell uptake promoter such as biotin was bound either to the polymer or to the therapeutic agent.
It is towards the further identification of means to enhance the ability of protein and other drugs to cross various barriers in the body, such as the intestinal mucosa and the blood-brain barrier, and to exhibit acceptable pharmacokinetic and other properties suitable for therapeutic use, that the present invention is directed.
The citation of any reference herein should not be construed as an admission that such reference is available as “Prior Art” to the instant application.